Phosphatidylinositol 4,5-bisphosphate (PIP2) is recognized as an essential modulator of transient receptor potential (TRP) channels. Specifically, it influences the vanilloid receptor I (TRPV1), a pain receptor activated by a wide range of stimuli, including the binding of phospholipids, such as PIP2. The primary PIP2-binding site in TRPV1 has been identified through advanced techniques, revealing that the PIP2 binds to a specific pocket composed of positively charged residues located predominantly within the proximal C-terminus region. Additionally, a conserved segment with positively charged amino acids, K431 and R432, situated at the beginning of TRPV1’s S1 transmembrane domain, has attracted considerable attention from the TRP research community. To date, our knowledge of this site’s function and the subsequent effects following PIP2 binding is still emerging. In this work, MD simulations were conducted using coarse-grained models to investigate the binding dynamics of PIP2 on both WT and various mutated forms of TRPV1 channels. Our findings indicate that the K431A and R432A mutations significantly reduce the frequency of PIP2 contacts, suggesting that these mutated residues are part of a “peripheral binding pocket.” This pocket seems to play a crucial role in facilitating the entry of PIP2 to the TRPV1 channel’s primary binding site. Furthermore, our research has shown that these highly conserved residues within the TRPV subfamily are also structurally conserved across other TRP subfamilies, such as TRPM and TRPC, a detail not evident from sequence alignment alone. Consequently, we propose the existence of a structurally conserved peripheral PIP2-binding site shared among the diverse members of the TRP family, which can be categorized into distinct subfamilies.

Protein–protein interactions (PPIs) play a central role in regulating cellular processes. However, the identification and characterization of senescence-associated PPIs remain challenging. In this study, we developed and evaluated an integrative methodology based on selective proteomics to identify PPIs associated with cellular senescence induced by TMPRSS11a. We started from isolated proteins that co-immunoprecipitated with TMPRSS11a and were subsequently identified by mass spectrometry. Building on this dataset, we implemented a workflow combining selective proteomics, structural bioinformatics, and experimental validation. Using this approach, we investigated the interaction between the transmembrane serine protease TMPRSS11a and the chaperone HSPA8. Structural bioinformatics analyses were performed to identify potential residues involved in the interaction interface, and the proximity of the TMPRSS11a–HSPA8 complex was evaluated using an in vitro proximity ligation assay. Our results provide evidence for an interaction between TMPRSS11a and HSPA8 and suggest its association with an enhanced senescence response. Overall, this study presents a workflow to investigate senescence-associated PPIs from proteomics-derived candidate proteins.